The apical surface of mammalian bladder urothelium is almost completely covered by urothelial plaques consisting of 2D crystals of 16-nm uroplakin particles. We have shown that these urothelial plaques are composed of four major uroplakins (UPIa, Ib, II and IIIa), and have studied the structure, function and disease implications of these major urothelial differentiation products. During the previous grant period, we made several key advances, including the generation of uroplakin knockout mice demonstrating that uroplakins contribute to the urothelial permeability barrier function, the development of a technique to biotinylate the apical surface of mouse bladder urothelium, and the discovery of several novel urothelial organelles that we believe play central roles in uroplakin trafficking and urothelial membrane expansion and repair. These findings allow us to test three important hypotheses. In Aim One, we will test the hypotheses that uroplakin endocytosis occurs via a novel, clathrin- and caveolin-independent process, and that uroplakin exocytosis plays a role in urothelial membrane expansion and repair. In Aim Two, we will test the hypothesis that uroplakins can function, in addition to contributing to the permeability barrier function, in enhancing the urothelial surface's ability to withstand excessive stretch thus preventing it from rupturing during bladder distention. In Aim Three, we will test the hypothesis that a novel urothelial organelle may play a key role in membrane expansion as well as repair. Our proposed studies wall utilize our uroplakin knockout mouse models, an in vivo tagging technique, and adenovirus to modulate, in vivo, key molecules in uroplakin trafficking. Our studies challenge some of the existing paradigms regarding urothelial membrane trafficking, address uroplakin functions in membrane stabilization, and elucidate the functional roles of novel urothelial organelles in membrane expansion and repair. Our results will enhance our understanding on how bladder urothelium functions under normal and over-stretched conditions, and have implications for important bladder conditions such as urinary tract obstruction and bacterial infection.